Methods, Systems and Apparatuses for Testing and Calibrating Fluorescent Scanners

ABSTRACT

Disclosed are calibration apparatuses for fluorescent microscopy instruments and methods of making and using them. Specifically, disclosed are calibration apparatuses with a fluorescent layer, such as photoresist, deposited on a substrate, with an optional layer of a reflective material, such as chrome. Illumination of the fluorescent and/or reflective layers, and detection and analysis of the resulting emissions allows evaluation of the instrument with respect to both reflective and fluorescent channels. Selection of appropriate fluorescent materials for the one or more fluorescent layers allows the evaluation of an instrument with respect to different fluorophores, as would be used with an instrument capable of two color detection. Inclusion of a reflective layer further allows the evaluation and calibration of all optical channels of an instrument, including the reflective channel and two or more fluorescent channels, with a single calibration apparatus for imaging criteria such as uniformity, contrast and emission signal strength.

RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication No. 61/546,872, filed Oct. 13, 2011 which is herebyincorporated herein in its entirety for all purposes.

FIELD OF THE INVENTION

The presently disclosed methods, systems, and apparatuses are related tofluorescence microscopy. Provided herein are calibration apparatuses andsystems and methods for using them for calibration of instrumentsutilized in fluorescence microscopy. The systems include a calibrationapparatus, an illumination source, an optical detector, and a computercomprising a computer readable medium storing computer-executable codefor controlling the system to illuminate the calibration apparatus,detect fluorescent emissions, and analyze the fluorescent emissions toidentify possible calibration requirements for optimal performance.

BACKGROUND OF THE INVENTION

Fluorescent microscopy is an important aspect in many fields, especiallybut not limited to applications in the life sciences. Many applicationsinvolving the detection, measurement and analysis of nucleic acids,proteins, antibodies, single or multiple cells, tissue samples, andother biological materials utilize one or more fluorescent labels fordetection of desired analytes. These applications include, for example,the use of nucleic acid microarrays for analysis of copy numbervariation, drug metabolism analysis, genome-wide or targeted genotyping,molecular cytogenetics, resequencing, gene profiling, gene expression,gene regulation, miRNA, whole-transcript expression and profiling, andother applications. Additional applications include the analysis ofproteins such as transcription factors or cytokines, nucleic acids atvarious levels and from various sources, including in situ with respectto single cells or tissue samples, and from cell extracts, tissuelysates, conditioned media, patient sera and plasma. Furtherapplications involve the use of fluorescent labels as a component withinvarious nucleic acid sequencing techniques, such as pyrosequencing,sequencing by ligation, unchained sequencing by ligation of DNAnanoballs, or sequencing by synthesis with reversible dye-terminators.

Methods, systems and apparatuses for the imaging of fluorescentlylabeled samples through the illumination and excitation of one or morelabels and the acquisition of one or more images of the correspondingfluorescent emissions are well known in the art. For example, U.S. Pat.Nos. 5,578,832; 5,834,758; 6,025,601; and 6,252,236 to Trulson et al.disclose methods, systems and apparatuses for generating electromagneticradiation of a particular wavelength, optics for focusing and directingthe radiation, optics for collecting responsive radiation fromfluorescently labeled samples, and assembling an image from the detectedresponsive radiation. Trulson et al. additionally disclose techniquesfor auto-focusing to maintain the sample within the focal plane of theexcitation light throughout the scanning process. U.S. Pat. Nos.5,631,734; 6,141,096; and 6,741,344 to Stern et al. disclose relatedmethods, systems and apparatuses for the detection of fluorescentlylabeled materials within a flow cell. Techniques utilizing galvanometermirrors as an aspect of the radiation component are discussed withinU.S. Pat. Nos. 5,981,956; 6,207,960; and 6,597,000 to Stern. Furthertechniques for increasing the field of view during scanning whilemaintaining a high scan speed and resolution are discussed within U.S.Pat. Nos. 6,185,030; 6,201,639; 6,335,824; and 7,312,919 to Overbeck.The

While the quality and accuracy of the resulting images of scannedfluorescent materials is dependent upon many factors, proper adjustmentof instruments for calibration and alignment is important to maintainingconsistent and accurate performance. For example, U.S. Pat. Nos.7,689,022; 7,871,812; and 7,983,467 to Weiner et al. discloseauto-focusing techniques to determine and maintain the best plane offocus for scanning Some of these techniques are based upon the use ofcalibration features, such as a chrome border, with the calibration andfocusing being performed using, for example, detection of the reflectedlight from the calibration features. Additional techniques utilizingpositional reference features to adjust and update a scanned image withrespect to expected and actual positions within the image are disclosedin U.S. Pat. No. 7,406,391 to Miles. However, the use of metal featuresis generally associated with calibration of the scanning instrument withrespect to alignment (e.g., exact positioning of the fluorescent targetwith respect to expected or optimal positions for illumination and/ordetection) and with respect to related aspects such as the calibrationof the relevant optical channel to be utilized for the alignment andpositioning determinations.

Fluorescence microscopy has been and continues to be a field ofsignificant research. Underlying principles of the excitation andemission of fluorophores, the operation of fluorescence microscopes,filtering options, potential light sources, and techniques to optimizefluorescence detection can be found in a variety of sources. (See, e.g.,Lichtman and Conchello, “Fluorescence microscopy,” Nature Methods, 2:910-919 (2005); Haustein and Schwille, “Trends in fluorescence imagingand related techniques to unravel biological information,” HFSP Journal,1(3): 169-180 (2007); Wolf, “Fundamentals of Fluorescence andFluorescence Microscopy, Methods in Cell Biology, 81: 63-91 (2007);Coling and Kachar, “Principles and Application of FluorescenceMicroscopy,” Current Protocols in Molecular Biology, 14: 14.10 (2001);Waters and Swedlow, “Interpreting Fluorescence Microscopy Images andMeasurements, Evaluating Techniques in Biomedical Research, Cell Press,pages 37-42 (2007)). The associated development of new excitationsources, detectors, imaging techniques, analysis techniques andfluorescent labeling chemistries is also widely described. (See, e.g.,Michalet et al., “The Power and Prospects of Fluorescence Microscopiesand Spectroscopies,” Annual Review of Biophysics and BiomolecularStructure, 32: 161-182 (2003); Wouters, “The physics and biology offluorescence microscopy in the life sciences,” Contemporary Physics,47(5): 239-255 (2007)). Optimization of the overall imaging systemrelated to fluorescence microscopy, such as utilization of light traps,improved fluorescent labels and image filtration routines, continue tobe explored. (See, e.g., Petty, “Fluorescence microscopy: Establishedand emerging methods, experimental strategies, and applications inimmunology,” Microscopy Research and Technique, 70(8): 687-709 (2007);Waters, “Accuracy and precision in quantitative fluorescence microscopy,The Journal of Cell Biology, 185(7): 1135-1148 (2009)). Many aspects offluorescence microscopy, however, remain difficult or time consuming tooptimize for particular configurations of instruments, fluorophores,imaging techniques, software and the other related aspects offluorescence microscopy. (See, e.g., Brown, “Fluorescencemicroscopy—avoiding the pitfalls,” Journal of Cell Science, 120:1703-1705 (2007); North, “Seeing is believing? A beginners' guide topractical pitfalls in image acquisition,” The Journal of Cell Biology,172(1): 9-18 (2006)). All of the above references are incorporatedherein in their entirety for all purposes.

Many fluorescent detection instruments utilize a plurality of opticalchannels, with one or more channels configured for the detection offluorescent emissions from one or more types of fluorophores. Previousapproaches regarding fluorescent channel calibration include U.S. Pat.Nos. 6,472,671 and 6,984,828 to Montagu, which discuss the use ofspecialized calibration tools, similar dimensionally to the actualobjects to be utilized with the instrument, and which possess afluorophore layer under a non-fluorescent layer which is etched to forma desired pattern. Subsequent excitation and detection of the resultantfluorescent signal through the non-fluorescent pattern allowscalibration of the one or more fluorescent channels of the relevantinstrument.

Accurate and precise evaluation, calibration, and testing of fluorescentchannels remain difficult in many respects. For example, creating andutilizing effective, cost-efficient fluorescent calibration targets thatexhibit emissions comparable in wavelength spectra and strength to thefluorescently labeled biological materials to be subsequently analyzedcontinues to be a need. Additionally, the creation of fluorescentcalibration targets that further possess characteristics that will beeffective in optimizing performance of the instrument and overallscanning system with respect to the precise requirements which willactually be utilized remains unfulfilled. Furthermore, techniques arealso needed to test and calibrate fluorescent channels while alsotesting and calibrating any associated reflective channels of theinstrument in a compact manner. This need is further complicated by therequirement to desirably maintain effectiveness for the testing of bothtypes of channels, and is further complicated for systems designed toevaluate and calibrate two or more different fluorescent channels, asmay be present in multi-color detection instruments.

SUMMARY OF THE INVENTION

Disclosed herein are methods of evaluating fluorescence detectionperformance by fluorescent microscopy instruments. Suitable calibrationtargets to be utilized within these methods, and methods for making thecalibration targets, are also disclosed. The calibration targets areuseful in calibrating fluorescent microscopy instruments for one or morefluorescent channels, and in some embodiments, one or more reflectivechannels. Thus, preferred embodiments utilize a single calibrationtarget with which to evaluate the performance of all optical aspects ofa fluorescent microscopy instrument.

The disclosed methods for evaluation of fluorescence detectionperformance by a fluorescent microscopy instrument involve theillumination of an appropriate calibration target for the excitation ofone or more fluorescent layers, detecting the resulting fluorescentemissions, and analyzing the fluorescent emissions to evaluate theperformance of the instrument. The analysis is often performed with acomputer software program with respect to pre-established criteria toguide the analysis of the emissions and to identify potential needs ofthe instrument for adjustment, calibration, or component replacement.

The one or more fluorescent layers comprise one or more fluorescentmaterials deposited on a substrate, with the materials being formed intoone or more patterns in many of the disclosed embodiments. Any suitablefluorescent material may be utilized, such as fluorescent photoresist,fluorescent glass, or polymers with fluorescent properties. In manyembodiments, the fluorescent materials are selected based upon theirsimilarity in excitation and/or emission characteristics to fluorophoresof interest with respect to the relevant instrument to be evaluated,such as commonly utilized fluorescent labels which are used to labelbiological materials that are subsequently analyzed by the instrument.

Some embodiments utilize methods and appropriate calibration targets forthe evaluation of instruments with multiple fluorescent channels andwhich will be for use with one or more different types of fluorescentlabels. To facilitate this, certain embodiments of calibration targetsutilize one or more fluorescent materials which possess differentemission properties with respect to different types of illumination,such as the use of different wavelength ranges for excitation. Theseembodiments allow the calibration of instruments with multiplefluorescent channels, such as those designed for use with a plurality offluorescent labels with different emission wavelength spectra for amulti-color analysis and/or applications involving multiplexing.

Many embodiments additionally include a reflective layer covering atleast a portion of one or more surfaces of the substrate. Materials suchas chromium or chromium oxide are utilized for the reflective layer inmany embodiments. The reflective layer is often patterned into one ormore fiducial markers for purposes such as positional alignment of thecalibration target, including translating, tilting, or rotating thecalibration target and/or instrument to obtain the optimal positioningfor scanning In certain embodiments, the one or more reflective layersand one or more fluorescent layers share the same pattern on thesubstrate, allowing the pattern to be formed within both layerssimultaneously, to simplify and optimize manufacturing. Certainembodiments will further package the calibration target before actualuse with the relevant instrument to be evaluated. For instance, acalibration target may be mounted upon a peg and placed on a sensorstrip or plate before being utilized with an instrument.

In many embodiments, a computer software program is used to analyze thedetected fluorescent emissions from the fluorescent layer and thereflection from the reflective layer of the calibration target. Theanalysis will often include a comparison of the detected data to one ormore pre-established criteria that reflect the desired optimal results,including uniformity, contrast and emission signal strength. Based uponthis analysis, software programs can identify potential aspects orcomponents of the instrument that require further attention, such ascalibration or replacement.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and following features will be more clearly appreciated fromthe following detailed description when taken in combination with theaccompany drawings. The drawings, however, are only intended to beillustrative of certain embodiments, and are not intended to belimiting.

FIG. 1 depicts a flow chart illustrating the process with which certainembodiments of the calibration target 140 are manufactured through steps210, 220, 230 and 240.

FIGS. 2(A)-2(C) illustrate three exemplary non-limiting embodiments ofcalibration targets from a vertical cross-sectional view. FIG. 2(A)depicts an embodiment with both a reflective layer 120 and a fluorescentlayer 130 on a substrate 110. FIG. 2(B) depicts an embodiment with onlya fluorescent layer 130 on a substrate 110. FIG. 2(C) depicts anembodiment with a spacing layer 150 deposited in-between reflectivelayer 120 and fluorescent layer 130 on a substrate 110.

FIGS. 3(A)-3(L) depict non-limiting examples of patterns which can becreated within the fluorescent layer 130, and in some embodiments,reflective layer 120, in forming calibration target 140.

FIGS. 4(A)-4(B) depict non-limiting examples of pattern combinations forfluorescent layer 130 which can be utilized within a single calibrationtarget 140.

FIGS. 5(A)-5(B) depict non-limiting examples of patterns which can becreated within reflective layer 120, and in some embodiments,fluorescent layer 130, in formatting fiducial markers on calibrationtarget 140. FIGS. 5(C)-5(D) depict examples of pattern combinationswhich can be utilized with a single calibration target 140.

FIGS. 6(A)-6(B) depict non-limiting examples of pattern combinationswhich possess both patterns for fluorescent layer 130 and patterns forfiducial markers from reflective layer 120.

FIG. 7 depicts a non-limiting example of a pattern suitable to create alarge quantity of calibration targets from a single substrate 110 with areflective layer 120 and fluorescent layer 130, from which can beseparated individual calibration targets 140 for subsequent use.

FIG. 8(A) depicts four pegs 805 mounted on a sensor strip 810, with thepotential for all or some of the pegs 805 to possess a calibrationtarget 140. FIG. 8(B) depicts ninety-six pegs 805 mounted on a sensorplate 815, with the potential for all or some of the pegs 805 to possessa calibration target 140.

FIG. 9 depicts a flow chart illustrating the process with which certainembodiments of calibration target 140 will be utilized with instrumentsfor calibration of one or more fluorescent channels, and optionally oneor more reflective channels, through use of calibration target 140.

FIGS. 10(A) and 10(B) depict images obtained by scanning calibrationtargets 140 with a GeneAtlas® Scanner (Affymetrix, Inc., Santa Clara,Calif.).

DETAILED DESCRIPTION

The present invention has many preferred embodiments and relies on manypatents, patent applications and other references for details known tothose of ordinary skill in the art to which the invention pertains.Therefore, when a reference, such as a patent, patent application, andother publication is cited or otherwise mentioned herein, it should beunderstood that the reference is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited.

The practice of embodiments of the present invention may also employconventional software methods and systems. Computer software productsutilized with embodiments of the present invention generally includecomputer readable medium having computer-executable instructions forperforming various steps directly or indirectly associated with aspectsof the present invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive (e.g., utilized locallyand/or over a network), flash memory, ROM/RAM, etc. The computerexecutable instructions may be written in a suitable computer languageor combination of several languages.

Throughout this disclosure, various aspects of the invention may bepresented in a range format. It should be understood that when adescription is provided in range format, this is merely for convenienceand brevity, and should not be construed as an inflexible limitation onthe scope of the invention. Accordingly, the description of a rangeshould be considered to have specifically disclosed all the possiblesub-ranges as well as individual numerical values within that range. Forexample, description of a range such as from 1 to 6 should be consideredto have specifically disclosed sub-ranges such as from 1 to 2, from 1 to2.5, from 1 to 3, from 1 to 3.5, from 1 to 4, from 1 to 4.5, from 1 to5, from 1 to 5.5, from 2 to 4, from 2 to 6, and from 3 to 6 for example,as well as individual numbers within that range, for example, 1, 1.5, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6. This applies regardless of thebreadth of the range.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a molecule”includes a plurality of such molecules, and the like.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art, are directed to the current application,and are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or patent application. Although any compositions,systems, and methods similar or equivalent to those described herein canbe used in the practice of the present invention, the preferredmaterials and methods are described herein. Accordingly, the terminologyused herein is for the purpose of describing particular embodimentsonly, is not intended to be limiting, and may be further supplemented byrelevant portions of this specification and by additional publicationsincorporated by reference.

The term “about” or “approximately” as used herein indicates the valueof a given quantity varies by +/−10% of the value, or optionally +/−5%of the value, or in some embodiments, by +/−1% of the value sodescribed.

A “calibration target” as used herein refers to an apparatus used withinan instrument system for the calibration of various aspects. Acalibration target may also be utilized for other purposes, such as thediagnosis of potential components of the instrument system which requirecalibration or replacement, quality control tests during manufacturingof the instrument, or during service repairs and maintenance forinstruments already in use. A calibration target includes a substrate,upon which is deposited one or more fluorescent layers, and with certainembodiments additionally including one or more reflective layers andoptionally one or more spacing layers, as each of those terms aredefined herein. A calibration target may be used directly with therelevant instrument system, or combined with additional componentsbecause of factors such as size and shape compatibility with theinstrument system.

A “fluorescent label” or “label” as used herein refer to a moiety thatfacilitates detection of a molecule through the principles offluorescence. Non-limiting examples of fluorescent labels includeorganic dyes, biological fluorophores, and quantum dots. Many labelswhich are suitable in the context of the various embodiments disclosedherein are commercially available, such as Alexa Fluor® dyes from LifeTechnologies Corporation (Carlsbad, Calif.) or DyLight® dyes from ThermoFisher Scientific, Inc. (Waltham, Mass.).

A “fluorescent layer” as used herein refers to one or more layers of oneor more materials suitable for placement on a substrate and whichpossess fluorescent properties suitable for use with an instrumentsystem utilized in fluorescent microscopy. The selection of materialsvaries from embodiment to embodiment and the desired fluorescentproperties of the resulting calibration target. Preferably, thematerial(s) are selected so that factors such as the emission wavelengthor wavelength ranges and the strength of the emissions are similar tothe fluorescent material that the instrument will subsequently analyzeafter calibration. Thus, many embodiments possess one or morefluorescent layers which possess properties similar to biologicalmaterials which are labeled with commonly available fluorescent labelsas discussed herein. The one or more fluorescent layers may be depositedupon either the substrate directly, upon a reflective layer, upon aspacing layer, or a combination of these depending on whether anyreflective or spacing layers have been deposited on the entirety of oneor more surfaces of the substrate, or merely on portions of one or moresurfaces. Additionally, the one or more fluorescent layers may bedeposited upon the entirely of one or more surfaces of the substrate orpreviously deposited layers on the substrate, or merely on portions. Theone or more fluorescent layers may be deposited through any suitabletechnique for the particular embodiment and the materials utilized. Manyembodiments involve the formation of one or more desired patterns withinthe fluorescent layer through a suitable technique. Embodimentsutilizing one or more reflective layers and/or one or more spacinglayers may also utilize these techniques in certain embodiments tosimultaneously create all the desired patterns within the calibrationtarget, as opposed to forming the patterns one layer at a time or only aportion of the patterns at a time. The patterns to be formed may be ofany suitable size, shape, separation and variety to aid calibration ofthe relevant instrument.

An “instrument” or “instrument system” as used interchangeably hereinrefer to any suitable instrument system designed for, or suitable foruse in, fluorescent microscopy. The instrument system is configured toilluminate a fluorescent molecule with light at a wavelength or range ofwavelengths that is suitable to excite the particular fluorophore atissue and cause a corresponding emission of light based upon theemission characteristics of the fluorophore. Often, these systems aredesigned or configurable for use with particular fluorescent moleculessuch that the emission wavelength or range of emission wavelengths areeasily distinguished from those utilized for excitation of thefluorescent molecules. The instrument system is also configured toobserve and/or acquire one or more images of the fluorescent emissions.Such instrument systems are often are associated with a computer and oneor more computer software programs, either directly (e.g., a built-inincorporated computer that is not designed to be separated from theinstrument) or indirectly (e.g., use of a desktop computer or otherremotely located computer) to operate the instrument for illuminationand detection, and to optionally acquire and save images of emissionsfor presentation to a user.

A “reflective layer” as used herein refers to one or more layers of oneor more materials suitable for placement on a substrate and which willreflect illumination for use by one or more reflective optical channelsof an instrument with which the calibration target will be used. Thesuitability of materials or combinations of materials depends uponfactors such as the type of illumination that will be utilized withrespect to the reflective layer, the desired properties of the reflectedlight (e.g., proportion of illumination that is reflected, wavelengthsof reflected light), the wavelength(s) which the instrument will utilizefrom the reflective light (e.g., if the instrument utilizes a filter),and other factors. The formation of the desired patterns with thereflective layer(s) may be done via any suitable technique. A reflectivelayer is not utilized within all embodiments of the calibration targetsdisclosed herein.

A “spacing layer” as used herein refers to a material or group ofmaterials within a calibration target that is optionally deposited onthe substrate or between layers that are deposited on the substrate(e.g., between a reflective layer and a fluorescent layer, between twodifferent reflective layers, between two different fluorescent layers).

A “substrate” as used herein refers to a material or group of materialshaving a rigid or semi-rigid surface or surfaces.

A “target” or “target material” or “analyte” or “sample” as usedinterchangeably herein refers to a molecule or group of molecules to bedetected by an instrument or instrument system, often through detectionof a fluorescent label directly or indirectly associated with themolecule or group of molecules. In many aspects the target is abiological material such as nucleic acids, polypeptides or antibodies.

II. Specific Embodiments

Various embodiments are contemplated herein relating to methods, systemsand apparatuses for the testing, evaluating and calibrating offluorescent channels of scanners, microscopes and other instruments thatutilize one or more fluorescent channels for the detection, measurement,and/or observation of fluorescent materials. Many embodiments aresuitable for use with fluorescent scanners and microscopes utilized inthe life sciences for the detection and measurement of fluorescentlylabeled biological materials. Additionally, many embodiments arecombined with, or are suitable for combination with the testing,evaluating, aligning and calibrating of one or more reflective channelsof scanners, microscopes and other instruments for purposes such aspositional alignment and calibration of the scanner or microscope withrespect to a fluorescently labeled target. It is to be understood thatthe following description, including any examples provided herein, isintended to be illustrative and not restricted to the followingembodiments. The examples and embodiments herein are for illustrativepurposes and serve as non-limiting examples, as many variations of theinvention will be apparent to those of skill in the art upon reviewingthe entire specification, including the appended claims, and the fullscope of equivalents to which the claims are entitled.

Creation of calibration target 140 for subsequent use in the calibrationof at least one fluorescent channel of a suitable instrument may follow,for example, the flowchart depicted in FIG. 1. Suitable instrumentsinclude microscopes and scanning devices designed to illuminate afluorophore with one or more wavelengths of light to cause the emissionof fluorescence, with subsequent observation and/or capture of thefluorescent emissions through an objective lens and/or camera. Suchinstruments are widely available commercially from, for example, NikonCorporation (Tokyo, Japan) or Olympus Corporation (Tokyo, Japan). Someembodiments are compatible for use with instruments configured for thedetection of fluorescent labels utilized in conjunction with instrumentsand systems for analysis of biological materials (e.g., nucleic acids,proteins), such as the GeneAtlas® Personal Microarray System, GeneTitan®Instrument or GeneTitan® Multi-Channel Instrument (Affymetrix, Inc.,Santa Clara, Calif.), the MS 200 Microarray Scanner (Roche NimbleGen,Inc., Madison, Wis.), the SureScan Microarray Scanner (AgilentTechnologies, Inc., Santa Clara, Calif.), or the HiScan™ System, iScanSystem, BeadXpress™ Reader, HiSeq™ Sequencing System, MiSeq™ System orGenome Analyer_(IIx) System (Illumina, Inc., San Diego, Calif.).

In step 210, a substrate 110 is provided. Substrate 110 comprises one ormore suitable materials or groups of materials having a rigid orsemi-rigid surface or surfaces. In many embodiments, at least onesurface of the substrate will be substantially flat (e.g., glass slides,silicon wafers), although in some embodiments the substrate may havevarious features, associated directly or indirectly, such as pegs,wells, pins, etched trenches, channels, flow cells, flow cell channels,raised regions or other features which may be structurally designed orinherent to the relevant material or materials. Non-limiting examples ofpotential substrate materials include glass, Si, Ge, GeAs, GaP, SiO₂,SiN₄, other silicon based materials, fused silica, fused quartz,polyvinylidene fluoride, polycarbonate, other polymers, and combinationsof these and other suitable materials known in the art. Suitablesubstrates may also be manufactured from two or more differentcomponents, with each component made out of one or more suitablematerials. Each component may or may not have different structuralfeatures and design aspects. A non-limiting example of a multi-componentsubstrate is a glass slide component within a silicon based frame.

Step 220 involves an optional step, where a reflective layer 120 may bedeposited upon substrate 110. Reflective layer 120 may also comprise oneor more layers of any suitable material or combination of materialswhich will reflect illumination for subsequent use by one or morereflective optical channels of an instrument with which the calibrationtarget will be used for purposes such as positional alignment andcalibration based upon, for example, reflectance from fiducial alignmentmarkers comprising reflective layer 120. The optimal material ormaterials may vary depending upon the embodiment, and the desiredstrength and wavelengths of the reflection signal based upon the type ofillumination used by the instrument. For example, instruments may employlight sources such as light emitting diodes, lamps (e.g., xenon arclamps, mercury-vapor lamps), or lasers (e.g., krypton, argon, coppervapor, Nd:YAG, helium neon). The power output of the illumination willvary greatly depending upon the particular system, and may be, forexample, from 1-25 milliwatts, such as 1, 2, 3, 4, 5, 7.5, 10, 15, 20,or 25 milliwatts. These non-limiting examples should not be construed asto be limiting, as various embodiments of calibration targets disclosedherein may be modified and adjusted for use with various instrumentsystems, including those with illumination power ranges within the aboverange (e.g., 12.5, 22.5 mW) or outside of the range (e.g., 0.75, 30, 50,100). Furthermore, the illumination may comprise light at variouswavelengths or ranges of wavelengths, as may be optimal for a particularinstrument or instrument system. Many embodiments of reflective layersare utilized with illumination wavelength(s) within the visible lightrange of electromagnetic radiation (e.g., from about 380 nanometers toabout 740 nanometers). Depending on the type of light source and theoptional use within the system of additional optical components (e.g.,filters, filter wheels, beam splitters), the illumination may comprise asingle wavelength (e.g., 420, 490, 520, 550, 650), a range ofwavelengths (e.g., 470-490, 525-550, 570-610), or combinations of these.

The selection of the material for reflective layer 120 will depend inlarge part upon the desired wavelength or wavelength range at which thereflectance will be used for subsequent calibration of the opticalchannel(s), auto-focusing, etc. For example, if the particular optics ofthe relevant instrument favor utilizing fiducials with high reflectancewith respect to light of 590 nanometers, then the material(s) with whichthose fiducials are made should possess a high reflectance with respectto such light, including other relevant factors such as the propertiesof the particular type of instrument, the properties of fluorescentlayer 130 (if fluorescent layer 130 will be deposited on top of relevantportions of reflective layer 120 that will be utilized by theinstrument), and the properties of any spacing layers 150 which areutilized within calibration target 140. Non-limiting examples ofmaterials suitable in certain embodiments include metals such aschromium, chromium oxide, silver, gold, nickel, aluminum, a materialwith a chrome plating layer, or mixtures or combinations of these (e.g.,a bottom layer of chromium with a top layer of chromium oxide).

In some embodiments, step 220 comprises depositing reflective layer 120onto substrate 110 using physical vapor deposition techniques. In apreferred embodiment, sputter deposition is utilized, but otherembodiments may employ alternative sputtering techniques, other physicalvapor deposition techniques, or other suitable methods employing, forexample, chemical vapor deposition, vacuum deposition, or, dependingupon the material, other techniques such as spin coating or dip coating.Reflective layer 120 may alternatively comprise a single layer of amaterial or group of materials, or more than two layers of materials orgroups of materials. For example, reflective layer 120 may be formed byusing sputter deposition to deposit a layer of chromium before a secondlayer of chromium oxide is deposited. Reflective layer 120 may bedeposited on an entire surface, on multiple surfaces, or selectedportions of one or more surfaces of substrate 110. The thickness ofreflective layer 120 will vary depending upon the embodiment and factorssuch as the material composition of substrate 110, the materialcomposition of reflective layer 120, the material composition offluorescent layer 130, the desired optical density of reflective layer120, the properties of the one or more optical channels of theinstrument for which reflective layer 120 will be utilized, thethickness of fluorescent layer 130 in relation to the thickness ofreflective layer 120, and other factors. Suitable thicknesses forreflective layer 120 in some embodiments range from 0.025 μm to 5.0 μm,such as 0.025 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.75μm, 1.0 μm, 1.5 μm, 2.5 μm, and 5.0 μm. Certain embodiments will utilizeother thicknesses within this range, such as 0.6 μm or 3.0 μm, or athickness outside of this range, such as 0.01 μm or 6.0 μm, if requiredfor the embodiment based upon, for example, the factors affectingthickness recited above. In a preferred embodiment, reflective layer 120comprises a chromium layer topped with a thin layer of chromium oxide toproduce a reflective layer 120 that is 0.1 μm thick with an opticaldensity of 3.0. In some embodiments, decreasing the thickness of thematerial(s) utilized for reflective layer 120 improves the resultingreflectance as the corresponding optical density decreases toward 1.

It should be noted that reflective layer 120 is an optional component,and not all embodiments will include a reflective layer 120. Alternativeembodiments of calibration target 140 may not utilize reflective aspectsfor calibration of reflective oriented channels of the instrument, withthe need and benefits of such calibration, auto-focusing and resultingpositional adjustments being performed by, for example, othercalibration devices or by reflective aspects which are utilized when thefluorescently labeled target material of interest is actually analyzed(e.g., a reflective layer or fiducial markers on a substrate with whichthe labeled target is associated). Still other embodiments may utilizealternate approaches such as built-in reflective layers or fiducialmarkers within substrate 110, or adding fiducial markers to calibrationtarget 140 at some later point (e.g., after fluorescent layer 130 hasbeen deposited, or after step 240 has been performed to form the desiredpattern of fluorescent layer 130). Non-limiting examples of possiblefiducial markers which can be added to calibration target 140 includethose illustrated in FIGS. 5(A) through 5(D). Still other embodimentsutilize one or more layers of a single material deposited on substrate110, the patterns of which are used for calibration of both reflectiveand fluorescent channels. Such materials must possess sufficientreflectivity for use with the reflective channel(s) while additionallybeing able to absorb and then fluoresce light at sufficient signallevels for use with the relevant instrument, and selection of suchmaterials will weigh heavily upon the characteristics of the instrument,such as its sensitivity and contrast abilities.

Step 230 involves depositing fluorescent layer 130. Fluorescent layer130 may comprise any suitable material or combination of materials,where at least one material possesses fluorescent properties suitablefor use in conjunction with the one or more optical channels of therelevant instrument which are utilized to detect fluorescent emissionsof the desired target material. The optimal material or materials willvary depending on the embodiment and various factors. For example, thenumber and characteristics of fluorescent emission wavelengths orwavelength ranges to be measured from the target after appropriateexcitation by the instrument system will guide the selection ofappropriate material(s). Other factors, such as the properties of theinstrument system (e.g., possible illumination wavelengths, detectionfilters to be utilized), and the number and types of differentinstruments with which a particular calibration target 140 will beutilized, will also guide the selection of appropriate material(s). Forexample, if the target possesses a fluorescent label of Cy3, fluorescentlayer 130 would desirably possess fluorescent emission properties in the550-600 nm range to provide a calibration target 140 with fluorescentproperties similar to that of the target. Furthermore, if the relevantinstrument is utilized with a plurality of dyes, such as utilizingfluorescein isothiocyanate in addition to Cy3, then fluorescent layer130 would desirably also possess fluorescent emission properties in the500-550 nm range as well to simulate the emission spectrum offluorescein isothiocyanate. Additionally, the one or more fluorescentlayers 130 of a calibration target 140 enable, in some embodiments, theuse of a single calibration target 140 for a multi-fluorescent channelinstrument. Other embodiments employ one or more fluorescent layers 130which are suitable, either separately or in combination, with multipleexcitation source and/or wavelength systems, and/or systems withmultiple excitation and/or emission filters.

Moreover, the resulting fluorescent emissions from fluorescent layer 130are desirably of strength comparable to the expected emissions from thelabeled target interest to further aid in a relevant calibration of theone or more fluorescent channels. In some embodiments, a plurality offluorescent layers 130 may be utilized, with at least some of the layerspossessing different fluorescent properties from the other layers.Certain embodiments employ from 2-5 distinct fluorescent layers. Thisrange should not be construed to be limiting, as many embodimentsutilize only one fluorescent layer, and other embodiments may optionallyemploy more than 5 layers. Alternatively, a first type of a fluorescentlayer 130 may be deposited for eventual use within select portions ofthe calibration target 140 while a second type of fluorescent layer 130with different properties is deposited for use within other portions ofcalibration target 140. Non-limiting examples of suitable materials forfluorescent layer 130, depending on the embodiment and the relevantinstrument, include photoresist materials possessing fluorescentproperties such as SU-8, fluorescent glass, various polymers withfluorescent properties such as poly(2-vinylnaphthalene), poly(2-naphthylmethacrylate), poly[N-(1-naphthyl)-N-phenylacrylamide], other polymerswith fluorescent properties, and combinations of these. Photoresistmaterial with fluorescent properties suitable for use within variousembodiments is available commercially from, for example, AZ ElectronicMaterials (Stockley Park, Middlesex, United Kingdom). Fluorescentpolymers suitable for use within certain embodiments are also availablecommercially from, for instance, Sigma-Aldrich Corporation (St. Louis,Mo.).

An advantage to utilizing photoresist materials with the desiredfluorescent characteristics is that the photoresist may often besubsequently patterned using standard lithography techniques. As withreflective layer 120, fluorescent layer 130 may be deposited by anymethod which is suitable for the particular material(s) used within theembodiment at issue, including, for example, spin coating, dip coating,chemical vapor deposition, physical vapor deposition, or vacuumdeposition. In preferred embodiments the photoresist material comprisingfluorescent layer 130 is deposited by spin coating.

If reflective layer 120 was deposited onto substrate 110 on one or moresurfaces, then fluorescent layer 130 may, in some embodiments, be atleast partially deposited on top of reflective layer 120 while leavingother areas of reflective layer 120 exposed. Other embodiments maydeposit fluorescent layer 130 entirely on top of reflective layer 120,which is illustrated in FIG. 2(A). If, however, a reflective layer 120was not deposited onto substrate 110, then fluorescent layer 130 may bedeposited directly onto substrate 110.

Furthermore, certain embodiments may optionally utilize one or morespacing layers 150 between substrate 110 and reflective layer 120,reflective layer 120 and fluorescent layer 130, or substrate 110 andfluorescent layer 130 (when a reflective layer 120 is not deposited).Thus, embodiments of calibration target 140 may utilize 0, 1, 2 or morespacing layers 150, with each layer comprising one or more layers of oneor more materials. Suitable thicknesses for spacing layer 150 in someembodiments range from 0.025 μm to 5.0 μm, such as 0.025 μm, 0.05 μm,0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.75 μm, 1.0 μm, 1.5 μm, 2.5 μm,and 5.0 μm. Certain embodiments will utilize other thicknesses withinthis range, such as 0.6 μm or 3.0 μm, or a thickness outside of thisrange, such as 0.01 μm or 6.0 μm. The one or more spacing layers 150 mayserve purposes such as aiding the manufacturing process (e.g.,facilitating the formation of the desired pattern of fluorescent layer130 within step 240), increasing efficiency of the resulting calibrationtarget 140 based on factors such as the optical characteristics of thematerials used within calibration target 140 or the optical detectionproperties of the relevant instrument, or in promoting adhesion of thedifferent layers (e.g., if the material(s) used within reflective layer120 do not naturally adhere to the material(s) used within fluorescentlayer 130). Some embodiments utilizing photoresist for fluorescent layer130 may utilize a spacing layer 150 of polyimide to aid in the adhesionof the polyimide to the reflective layer 120. The purpose of spacinglayer 150 in a particular embodiment will guide the selection of anappropriate material. For instance, if a spacing layer 150 is utilizedbetween reflective layer 120 and fluorescent layer 130, a material forspacing layer 150 may be selected which is substantially opticallytransparent in order to avoid a substantial reduction in the reflectanceof reflective layer 120 with respect to the one or more reflectivechannels of the instrument. Such an embodiment with spacing layer 150 isillustrated in FIG. 2(C).

Step 240 involves the formation of a desired pattern of fluorescentlayer 130. Embodiments utilizing a reflective layer 120 may also have apattern formed within reflective layer 120 during step 240 as well, oralternatively utilize two or more steps to create the patterns withinfluorescent layer 130 and reflective layer 120. The formation of thedesired pattern may be achieved through any suitable technique, such asphotolithography, electron beam lithography, optical masklesslithography such as direct laser writing or interference lithography,ion beam lithography, destructive scanning probe lithography, plasmaetching, wet etching, or other techniques known in the art. Certainembodiments utilizing a fluorescent layer 130 comprising photoresist anda reflective layer 120 comprising chromium may preferably utilizecontact lithography.

The pattern may be any appropriate pattern for facilitating calibrationof the one or more fluorescent channels of the instrument and/orreflective channels of the instrument. Non-limiting examples of possiblepatterns are illustrated in FIGS. 3(A) through 3(L). Various embodimentsmay utilize a plurality of patterns within a single calibration target140 with certain patterns repeated in different locations, repeated indifferent locations with alterations such as changes to size, shape,distribution and/or rotation, and/or in combination with other distinctpatterns. Non-limiting examples of potential combinations of patternsare illustrated in FIGS. 4(A) and 4(B). Preferably, the patterns will beof a design with characteristics such as pattern variety, size, shape,and separation that aids in providing the relevant instrument with acalibration target 140 which has similar properties to the eventualfluorescently labeled material to be analyzed.

The formation of appropriate patterns allows calibration of theinstrument in a manner that produces more accurate results of thematerial of interest. For example, if the expected fluorescent emissionsto be captured by the instrument are 5 μm in diameter, a relevantembodiment of calibration target 140 would desirably possess at leastsome patterns within fluorescent layer 130 with structural featuresapproximately 5 μm in diameter. Similarly, if the actual fluorescentlabels are expected to be separated from each other by, for instance, 2μm, the relevant embodiment of calibration target 140 would preferablyhave at least some structural features within fluorescent layer 130which are separated by approximately 2 μm.

Many embodiments which utilize a reflective layer 120 will have thedesired pattern(s) formed during step 240 in both the fluorescent layer130 and the reflective layer 120. Formation of a calibration target 140in this manner potentially assists in producing a dark (low signal)background if substrate 110 is also properly selected. With respect tothe created pattern(s) within fluorescent layer 130, a dark backgroundfor corresponding fluorescent channels of the instrument will beprovided if substrate 110 is selected from materials such that anyfluorescent emissions at the relevant wavelengths are significantlylower than the fluorescent emissions from the fluorescent pattern withinfluorescent layer 130. Furthermore, appropriate selection of materialsfor substrate 110 can also ensure that a low background signal isproduced when reflective layer 120 is utilized with the one or morereflective channels of the instrument.

Creating the pattern in both the fluorescent layer 130 and reflectivelayer 120 may provide additional benefits in certain embodiments aswell. For example, certain techniques utilized to create the patternsmay not be completely precise with respect to the depth of the materialremoved from the layers deposited on substrate 110, such as fluorescentlayer 130, reflective layer 120 and any spacing layers 150. Thus, if theentirety of fluorescent layer 130 and/or reflective layer 120 is notcompletely removed from desired areas of the pattern, any residue fromeach may contribute to undesirable background signal. Therefore, certainembodiments will employ the desired pattern creation technique to createthe patterns at a depth that extends slightly beyond the expectedcombined thickness of reflective layer 120, fluorescent layer 130 andany spacing layers 150 which have been utilized. This will often result,depending on the technique, in a slight removal of material fromsubstrate 110, but provides the benefit of facilitating a darkerbackground for the resulting calibration through the completeelimination of reflective layer 120, fluorescent layer 130 and anyspacing layers 150 within the desired areas of substrate 110.Accordingly, design of calibration targets 140 which are compatible withdifferent fluorescent excitation/emission spectra, various instruments,and/or the inclusion of a reflective layer can greatly facilitate thecomplete calibration of all optical channels and related aspects of therelevant instrument with a limited number of types of calibration target140, or more preferably with a single type of calibration target 140.

Embodiments which combine a reflective layer 120 with a fluorescentlayer 130 may create additional patterns for calibration target 140 inaddition to the patterns created for use with the fluorescent layer 130and any relevant fluorescent channels of the instrument. For example,functions such as positional alignment and calibration of opticalcomponents, auto-focusing during illumination and/or imaging, andpositional adjustments to the labeled material (or the substrate withwhich the labeled material is associated) often utilize one or morefiducial markers, such as borders or shapes placed in known locations(e.g., corners). Non-limiting examples include the illustrative shapesin FIGS. 5(A) and 5(B). Certain embodiments may use one type offiducial, or combinations of types, as illustrated in FIGS. 5(C) and5(D) respectively. Thus, the possible pattern combinations discussedabove and illustrated in FIGS. 4(A) and 4(B) respectively can becombined with fiducial patterns to create a reflective layer 120 and afluorescent layer 130 on substrate 110 as depicted in FIGS. 6(A) and6(B).

In such combination embodiments, it is generally preferred to simplifythe manufacturing process by forming the desired pattern for both thefluorescent calibration and the reflective calibration at the same timewithin step 240. Thus, the resulting structural features on substrate110 would have both a reflective layer 120 with a fluorescent layer 130positioned on top of reflective layer 120. While such a manufacturingprocess is more efficient, it is important to consider this structuralrelationship when designing such an embodiment, as fluorescent layer 130(as well as a possible spacing layer 150) must not reduce thereflectance of reflective layer 120 to a degree where effective use ofthe fiducial markers is lost given the optical characteristics of therelevant instrument. Furthermore, the reflectance from reflective layer120 must not significantly disrupt the detected fluorescence fromfluorescent layer 130 at the relevant wavelengths during excitation offluorescent layer 130 to a degree which interferes in accuratecalibration of the one or more fluorescent channels of the instrument.Proper consideration of these factors, however, allows embodiments whereall desired layers can be deposited upon substrate 110, followed by theformation of the desired patterns for reflective and fluorescent use ina single step.

The above processes can be expanded, as illustrated in FIG. 7, to createa large quantity of calibration targets 140. Depending upon the desirednumber of calibration targets and the manufacturing process utilized,dozens, hundreds, or thousands of calibration targets can be createdsimultaneously. For example, FIG. 7 depicts 289 calibration targets. Thequantity, of course, can be adapted to whichever format may be mostconvenient based upon factors such as the exact size and format of thecalibration targets 140, the particular manufacturing techniquesinvolved, etc. After manufacturing, these calibration targets 140 canthen be separated from one another for use with various instruments.

After separation, some calibration targets 140 may be suitable for usedirectly with the relevant instrument. For example, if substrate 110 isa glass slide and the relevant instrument analyzes fluorescently labeledmaterials on glass slides, then corresponding embodiments of calibrationtarget 140 may simply be used directly with the instrument. Othercalibration targets 140, however, may need to be combined with othercomponents before use with an instrument because of, for example, sizeand shape compatibility with the instrument or physical or chemicalhandling concerns. A non-limiting example of such a combination is useof calibration target 140 with a GeneChip® Array Cartridge (Affymetrix,Inc., Santa Clara, Calif.), non-limiting variations of which aredescribed within U.S. Pat. Nos. 5,945,344; 6,140,044; 6,399,365;6,551,817; and 6,733,977 to Besemer et al.

Calibration target 140 may also be mounted upon protrusions such as pegsfor subsequent use with the instrument. U.S. Pat. No. 6,660,233 toCoassin et al. describe the detection of fluorescently labeled targetson pegs, and in some embodiments calibration target 140 is suitable foruse in such systems. Use of multiple pegs within a single sensor plateor strip is described in U.S. patent application Ser. Nos. 11/243,621and 11/347,654 to Yamamoto et al. Additional related applicationsinclude U.S. patent application Ser. Nos. 12/963,593 and 13/157,268 toShirazi et al. Such plates and strips allow, for example, for moresamples to be analyzed during a given time period. As illustrativenon-limiting examples, FIG. 8(A) depicts an embodiment with a sensorstrip 810 of four pegs 805 while FIG. 8(B) depicts an embodiment with asensor plate 815 of ninety-six pegs 805. While a calibration target 140may be placed on each and every peg within a particular plate or strip,such placement is not normally necessary for a precise and accuratecalibration for many instruments. For example, certain embodiments ofstrips 810 may utilize one or two calibration targets 140 while certainembodiments of plates 815 may utilize, for instance, 1-10 calibrationtargets 140. Certain embodiments of plates 815 place calibration targets140 at the borders of plate 815, such as at the four corner pegs ofplate 815, with one or more calibration targets 140 placed morecentrally within the assembly of pegs 805, or a combination of the twoplacements. The pegs of the strips 810 or plates 815 which do notpossess calibration targets 140 may simply be left blank if calibrationtarget 140 will only be used to calibrate the relevant instruments.

In other embodiments, strips 810 or plates 815 utilize calibrationtargets 140 in combination with the actual fluorescently labeledmaterial of interest. For example, some embodiments utilize calibrationtargets 140 in combination with nucleic acid probe arrays designed tohybridize with a nucleic acid sample. Use of calibration targets 140 inthis manner facilitates calibration of the relevant fluorescent channelsof the instrument each time a particular fluorescently labeled materialof interest is analyzed. While such continual calibration may notnormally be required for many instruments, certain assays or experimentswhich require especially consistent and accurate results with respect toexpected performance baselines can be improved by the use of one or morecalibration targets 140 in combination with the fluorescently labeledmaterials of interest.

Subsequent use of calibration target 140 will vary depending on theembodiment and the relevant instrument, but certain embodiments mayfollow the flowchart depicted in FIG. 9. In step 910, calibration target140 is illuminated by the relevant instrument to be calibrated. Thus,the type of illumination will depend on the characteristics of theinstrument with respect to factors such as the source type (e.g., lightemitting diodes, lasers, lamps), duration of illumination, the number ofsources, the use of various optical components affecting the outputillumination (e.g., filters, beam splitters) and other factors known inthe art. For more effective calibration, the characteristics ofillumination in step 910 will be tailored with respect to factors suchas the properties of the particular type of calibration target 140, anddesirably the fluorescently labeled material that will be subsequentlyanalyzed by the instrument after calibration. In many embodiments, theillumination and overall instrument will be controlled, at least inpart, by a computer, often following instructions stored within computercode and/or directed through input commands. In some applications, theillumination may be repeated one or more times, at the same or differentwavelengths, depending upon the properties of the calibration target(e.g., whether the calibration target possesses a reflective layer 120or fiducial markers, whether the calibration target is designed tosimulate a plurality of different fluorophores) and the instrument(e.g., whether multiple different fluorescent channels are utilized).

Step 920 comprises the observation of calibration target 140 afterillumination, and/or the acquisition of one or more images of at least aportion of calibration target 140, or in some embodiments the entiretyof calibration target 140. Observation and acquisition of images canalso be achieved by any suitable means known in the art (e.g.,fluorescent microscopes including confocal, inverted, and total internalreflection fluorescence microscopes, a charge-coupled device (CCD) orvariants such as electron-multiplying CCD or frame transfer CCD, or by acomplementary metal-oxide-semiconductor (CMOS) approach such as anactive pixel sensor), and will depend upon the embodiment and theparticular instrument. If one or more images are acquired, manyembodiments will store the images, for example on an associated computeron a computer-readable medium (e.g., hard drive, USB drive) or stored onan associated network server.

Depending upon the embodiment, steps 910 and 920 may be repeated one ormore times. For example, the instrument may illuminate the reflectivelayer 120 as positioned within fiducials (e.g., the fiducialsillustrated in FIGS. 5(A)-5(D)) for purposes such as auto-focusingand/or calibration of the one or more reflective channels of theinstrument before subsequent illumination of one or more patterns withinfluorescent layer 130 and calibration of the one or more fluorescentchannels. In the non-limiting examples illustrated in FIGS. 6(A)-6(B),this may involve illumination and image acquisition of the four fiducialmarkers in the four corners of calibration target 140 beforeillumination and acquisition of the nine sets of patterns within thecenter of calibration target 140. Furthermore, many embodiments ofcalibration target 140 are optimally used with different illuminationwavelengths or ranges of wavelengths with respect to the one or morereflective layers 120 and the one or more fluorescent layers 130. Thus,in addition to repeating step 910 for illumination of different portionsof calibration target 140, other embodiments may repeat the illuminationat different wavelengths while illuminating all or part of calibrationtarget 140. Within the embodiments utilizing one or more fluorescentlayers 130 for calibration of the instrument with respect to a pluralityof fluorescent channels at one or more wavelengths or ranges ofwavelengths (e.g., for calibration of an instrument that will beutilized with two or more types of fluorescent labels, with each typepossessing a range of wavelengths for excitation and/or emission thatare at least partially distinct from one another, or that are entirelynon-overlapping inherently or after the use of appropriate filters), thefluorescent layers 130 may require multiple illuminations from one ormore light sources at one or more wavelengths or ranges of wavelengths(e.g., use of different light emitting diodes at different wavelengths,use of different excitation filters with a lamp).

Step 930 comprises analysis of the observations or the acquired image(s)of calibration target 140. In many applications, a software program willanalyze the observations and/or images for various aspects including,for example, uniformity, contrast, and emission signal strength, oftenby comparing these aspects quantitatively to pre-established criteria.The pre-established criteria can be stored on a computer for access bythe program, and in certain applications is updated or is otherwisecapable of editing by an automatic or manual process. The comparison caninclude, for example, whether a particular aspect of the acquired datais within a certain percentage range of the pre-established criteria.Certain applications also provide pass/fail calls based upon theseaspects and criteria with respect to the general calibration analysis(e.g., instrument is in need of further calibration) or with respect tocertain aspects (e.g., a lower than expected level of emissions is beingdetected for a particular illumination wavelength and a specificdetection filter). The pass/fall call determinations may subsequently bestored in memory for subsequent use and/or presentation to a user. Theapplication may be a component of the routine analysis platform for theinstrument, or may be a supplemental application utilized, for example,during manufacturing quality control tests or during service maintenanceand repair tests. As with steps 910 and 920, certain instruments andapplications may repeat the analysis within step 930 one or more timesafter one or more subsequent repetitions of steps 910 and/or 920 (e.g.,repeating the analysis for the patterns within fluorescent layer 130after analysis of the fiducial markers of reflective layer 120).

Certain embodiments may additionally employ step 940, where theinstrument is calibrated based upon the analysis of step 930. Dependingupon the instrument and controlling software, certain applications mayadditionally utilize the analysis results to adjust the aspects of theprocess, such as positional adjustments (e.g., translating, rotating,tilting the instrument and/or calibration target 140 for more optimalperformance), illumination adjustments (e.g., adjusting the focus,duration or wavelength of illumination), or adjustments to theobservation of fluorescent emissions or the image acquisitions of theemissions (e.g., filter or focusing adjustments, adjustments to therelevant image capture device such as a CCD or CMOS device).Furthermore, certain applications will additionally analyze any failcalls resulting from step 930 to provide additional diagnostics andguidance as to required adjustments or component replacements basedupon, for example, substantial discrepancies between the observations oracquired image(s) and the pre-established criteria. In an additionalembodiment, these applications can also alert a user through, forexample, a graphical user interface, e-mail, text message, or instantmessage, that one or more components require further testing,calibration, and/or replacement, as depicted in step 950.

III. Examples

The images shown in FIGS. 10(A) and 10(B) were obtained from use of acalibration target 140 possessing a fluorescent layer 130 with patternssuch as those illustrated within FIGS. 4(A) and 4(B). The calibrationtarget 140 comprised a substrate 110 of fused silica, a reflective layer120 of chromium with a thickness of 0.1 μm, and a fluorescent layer ofAZ® photoresist (AZ Electronic Materials, Stockley Park, Middlesex,United Kingdom) with a thickness of 0.5 μm. The resulting calibrationtargets 140 were mounted on pegs, and scanned with a GeneAtlas® Scanner(Affymetrix, Inc., Santa Clara, Calif.) utilizing a 530 nm lightemitting diode for illumination and a CCD camera for imaging, with aresolution of 1 μm and a digital resolution of 12 bits. The images wereobtained after an exposure time of 3000 ms for the calibration target140 in FIG. 10(A) and 2000 ms for the calibration target 140 in FIG.10(B), while the fluorescence detection wavelength for both images was590 nm±20 nm.

It is to be understood that the above description, including anyexamples provided herein, is intended to be illustrative and notrestrictive. Many variations of the invention will be apparent to thoseof skill in the art upon reviewing the above description. The scope ofthe invention should be determined with reference to the appendedclaims, along with the full scope of equivalents to which such claimsare entitled. All cited references, including patent and non-patentliterature, are incorporated herein by reference in their entirety forall purposes.

1. A method of evaluating fluorescence detection performance by afluorescent microscopy instrument, the method comprising: illuminating acalibration target, wherein the calibration target comprises a substrateand a fluorescent layer, and wherein the fluorescent layer isilluminated with light configured to produce fluorescent emissions;detecting the fluorescent emissions with a fluorescent microscopyinstrument; and analyzing the fluorescent emissions with a computer,wherein the computer comprises a computer software program, and whereinanalyzing comprises comparing the fluorescent emissions to one or morepre-established criteria.
 2. The method of claim 1, wherein thefluorescent layer comprises one or more fluorescent materials formedinto one or more patterns.
 3. The method of claim 2, wherein thefluorescent layer comprises photoresist.
 4. The method of claim 1,wherein the calibration target is utilized to evaluate a plurality ofoptical channels within the fluorescent microscopy instrument.
 5. Themethod of claim 4, wherein the plurality of optical channels comprisesat least two fluorescent channels.
 6. The method of claim 5, whereinilluminating the calibration target comprises a first illumination withlight comprising a first set of one or more excitation wavelengths and asecond illumination with light comprising a second set of one or moreexcitation wavelengths, and wherein the first set of one or moreexcitation wavelengths and the second set of one or more excitationwavelengths are at least partially non-overlapping.
 7. The method ofclaim 6, wherein the first illumination produces a first set ofemissions from the fluorescent layer and the second illuminationproduces a second set of emissions from the fluorescent layer, andwherein the computer analyzes the first set of emissions to evaluate afirst fluorescent channel and the second set of emissions to evaluate asecond fluorescent channel.
 8. The method of claim 7, wherein the firstset of emissions and the second set of emissions comprise differentemission spectra.
 9. The method of claim 7, wherein the fluorescentlayer comprises one or more fluorescent materials such that the firstset of emissions possess emission properties similar to a first type offluorophore and the second set of emission possess emission propertiessimilar to a second type of fluorophore.
 10. The method of claim 2,wherein the fluorescent layer comprises a first fluorescent material anda second fluorescent material, and wherein the first fluorescentmaterial and second fluorescent material possess different excitationand emission properties.
 11. The method of claim 1, wherein thecalibration target additionally comprises a reflective layer, andwherein the method additionally comprises: illuminating the calibrationtarget, wherein the reflective layer is illuminated with lightconfigured to produce a reflection detected by the fluorescentmicroscopy instrument; and adjusting the positional alignment of thecalibration target with respect to the fluorescent microscopy instrumentbased upon detected reflection from the reflective layer.
 12. The methodof claim 11, wherein the reflective layer comprises one or morereflective materials formed into one or more patterns.
 13. The method ofclaim 11, wherein the reflective layer comprises one or more reflectivematerials formed into one or more patterns, and wherein the fluorescentlayer comprises one or more fluorescent materials formed into one ormore patterns identical to the one or more patterns of the reflectivematerials.
 14. The method of claim 12, wherein one or more patterns ofthe one or more reflective materials are used by the fluorescentmicroscopy instrument as fiducial markers for adjusting the positionalalignment.
 15. The method of claim 1, wherein the one or morepre-established criteria are quantitative values.
 16. The method ofclaim 1, wherein the quantitative values represent characteristics ofthe calibration target selected from the group consisting of uniformity,contrast and emission signal strength.
 17. The method of claim 1,wherein analyzing additionally comprises determining whether thefluorescent emissions satisfy each of the pre-established criteria.18-35. (canceled)